Cubosomes of dapsone enhanced permeation across the skin

Accepted Manuscript Cubosomes of dapsone enhanced permeation across the skin Radhakrishnan Nithya, Prince Jerold, Karthik Siram PII: S1773-2247(18)30399-X DOI: 10.1016/j.jddst.2018.09.002 Reference: JDDST 763 To appear in: Journal of Drug Delivery Science and Technology Received Date: 20 April 2018 Revised Date: 13 August 2018 Accepted Date: 2 September 2018 Please cite this article as: R. Nithya, P. Jerold, K. Siram, Cubosomes of dapsone enhanced permeation across the skin, Journal of Drug Delivery Science and Technology (2018), doi: 10.1016/ j.jddst.2018.09.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT AC C EP TE D M AN US C RI PT Graphical abstract ACCEPTED MANUSCRIPT Cubosomes of dapsone enhanced permeation across the skin Radhakrishnan Nithya1*, Prince Jerold1, Karthik Siram1 Department of Pharmaceutics, PSG College of Pharmacy, Peelamedu, Coimbatore 641004, Tamilnadu, India R.Nithya, AC C Assistant professor, EP * Address for Correspondence: TE D M AN US C RI PT 1 PSG College of Pharmacy, Coimbatore, India Email : [email protected] Mobile: +91978887041 1 ACCEPTED MANUSCRIPT Abstract: The current research work was performed with an objective to deliver dapsone across the skin using cubosomes. Dapsone loaded cubosomes (DC) were prepared by ultrasonication of aqueous dispersion containing cubic gel matrix of glyceryl monooleate (GMO) and poloxamer 407. The RI PT formulations were characterized by their particle size, surface morphology, zeta potential, entrapment efficiency and in vitro release in pH 7.4 phosphate buffer saline (PBS) containing 1% tween 80. In vitro permeation study using pig ear skin was performed for DC, dapsone solubilised in pH 7.4 PBS (dapsone-PBS), and a marketed product, to assess if encapsulation of dapsone in M AN US C cubosomes enhanced the permeation across the epidermis. The neutrally charged cubic shaped particles were in the size range of 39.4 ± 3.6 nm to 231.9 ± 7.1 nm. Dapsone encapsulated in cubic shaped lipid structures showed highest transdermal flux value (71.28 ± 4.65 µg/cm2/h) when compared to marketed formulation (55.28 ± 2.13 µg/cm2/h) and dapsone-PBS (45.44 ± 3.09 µg/cm2/h). The result indicates that DC are a good option to enhance permeation across the D epidermal layers of the skin. AC C EP TE Keywords: Cubosomes; dapsone; glyceryl monooleate; poloxamer 407; transdermal flux 2 ACCEPTED MANUSCRIPT 1. Introduction Lipid-based drug delivery systems have gained a lot of attention in the past few decades owing to different reasons such their ability to entrap both lipophilic and hydrophilic drugs, biocompatibility, biodegradability, and economic feasibility [1,2]. They can enhance the delivery of drugs to blood, RI PT brain, cancerous cells, lymphatic organs, and across the skin [3–7]. Considerable efforts in lipid based drug delivery systems have resulted in the development of different kinds of carriers including liposomes, ethosomes, phytosomes, solid lipid nanoparticles, nanostructured lipid, M AN US C cubosomes, etc, each of them possessing peculiar advantages and disadvantages [3,7–10]. In recent times, the application of cubosomes has been increasing owing to its specific shape, stability, and biocompatibility [11]. Cubosomes are crystalline isotropic lipidic nanoparticles stabilized by poloxamers [10,11]. They are sterically stabilized inverse bi-continuous cubic phases of lipids that are colloidally and thermodynamically stable. They are generally formed by either dispersion or fragmentation of the D cubic phases of gels in aqueous environment. Due to their high internal surface area per unit volume TE (~400 m2/g) and a three dimensional structure with hydrophilic and hydrophobic domains, they can entrap hydrophilic, hydrophobic and amphiphilic substances efficiently [12,13]. Additionally, they EP can release the entrapped drugs in a sustained fashion. They are generally prepared using AC C unsaturated monoglycerides (e.g. monoolein) and stabilized by nonionic surfactants. Due to the similarity between the inner structure of cubosomes and the stratum corneum, cubosomes facilitate entry of drugs across the epidermis of the skin [14–16]. Dapsone is a sulfone class antibiotic and anti-inflammatory drug used in treating acne, leprosy, Kaposi’s sarcoma, epidermolysis bullosa acquisita, dermatitis herpetiformis, Behcet’s disease, and systemic lupus erythematosus [17,18]. But, due to low bioavailability and adverse effects including peripheral neuropathy, hemolytic anemia, nausea and headache, its application in clinical set up is hindered. These side effects arise due to the production of dapsone hydroxylamine by acetylation 3 ACCEPTED MANUSCRIPT and enzymatic hydroxylation in the liver [19]. So, delivery of dapsone across the skin directly to the site of inflammation or infection would be a viable option in countering the above mentioned issues. A series of carriers including polymer-lipid-polymer hybrid nanoparticles, nanoemulsion, and hydrogel containing lipid-core nanocapsules have been prepared to enhance the permeation of RI PT dapsone across the skin till date [17,20,21]. However, there are no available reports regarding the application of cubosomes to enhance the permeation across the skin. Through this study, an attempt was made for the first time to explore the ability of cubosomes to enhance the permeation of M AN US C dapsone across the skin. 2. Materials and methods 2.1 Materials Dapsone was obtained as a gift sample from Ameya Pharmaceuticals and Chemicals Pvt Ltd, Mumbai, India. GMO was obtained as a gift sample from Mohini Organic Pvt ltd., Mumbai, India. Poloxamer 407 was procured from Fisher Scientific India Ltd., Mumbai, India. All the other D chemicals and reagents used in the study were of analytical grade. The marketed product used in the EP 2.2 Preparation of cubosomes TE work corresponds to Acnedap (Dapsone gel 5%), Cipla Limited, India. AC C Different batches of DC (20 ml) were prepared by ultrasonication of cubic gels of GMO and poloxamer 407 in aqueous environment. Briefly, GMO and poloxamer 407 were melted at 60 °C and dapsone (0.25%) was added to this melted mixture to form a clear homogenous lipid phase. Water (2 ml) preheated at 60°C was gradually added and the mixture was equilibrated for 48 h at room temperature (25 °C) to form a clear cubic phase gel. The cubic phase gel was hydrated with approximately 18 ml water and disrupted under mechanical stirring at 1000 rpm. The crude dispersion of the gel was fragmented for 15 min using a probe sonicator (Sonic Vibra Cell) at 15 s on cycle and 5 s off cycle to form a milky coarse dispersion of DC [22]. The composition of various batches of DC was mentioned in Table 1. 4 ACCEPTED MANUSCRIPT 2.3 Measurement of particle size and polydispersity index (PDI) The average particle size and PDI of DC was measured by Malvern Zetasizer (Nano ZS90, Malvern instruments) at 25 °C. The samples were kept in polystyrene cuvette and the readings were measured at a fixed angle [4]. RI PT 2.4 Measurement of zeta potential The zeta potential of DC was measured by Malvern Zetasizer (Nano ZS90, Malvern instruments) at 25 °C. The samples were kept in the polystyrene cuvette and a zeta dip cell was used to measure the M AN US C zeta potential [4]. 2.5 Visualization of DC by scanning electron microscopy (SEM) and atomic force microscopy (AFM) For visualizing cubosomes under SEM, 10 µl of DC was uniformly spread on a glass slide and allowed to dry at room temperature. After gold coating the sample with a Polaron E5100 gold D sputter coater, the morphology of DC was observed under a Philips 505 electron microscope at an TE accelerating voltage of 20 kV. For visualizing cubosomes under AFM, 10 µl of DC was adsorbed on the surface of a silicon wafer EP and allowed to dry at room temperature. The morphology of the DC was observed using Multimode AC C Scanning Probe Microscope (NTMDT, NTEGRA Prima, Russia) in semi-contact mode. 2.6 Entrapment efficiency The entrapment efficiency of DC was calculated using ultracentrifugation technique [23]. Briefly, one ml of the formulation was centrifuged at a speed of 30,000 rpm (107,662 × g) for 1 h at 4 °C. The supernatant was collected and the amount of dapsone was determined by ultraviolet visible spectroscopy at 293 nm. Entrapment efficiency was calculated using the following equation: 5 ACCEPTED MANUSCRIPT Encapsulation efficiency % = amount of drug added during preparation − amount of free drug in the supernatant × 100 amount of drug added during preparation 2.7 In vitro drug release study In vitro release studies of dapsone from DC was performed using a dialysis membrane with a RI PT molecular weight cut off of 12,000- 14,000 Da (HiMedia Laboratories) using pH 7.4 phosphate buffer saline (PBS) containing 1% tween 80 as the buffer solution [24]. One ml of DC solution was instilled in the dialysis bag and the ends were firmly sealed using dialysis clamps. This sealed M AN US C membrane was suspended in a beaker containing 250 ml of buffer solution maintained at 37 ± 1 °C and stirred at 50 rpm using a magnetic stirrer. At different time intervals (0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h), 1 ml of buffer solution was withdrawn and the same volume of fresh dissolution medium was replenished. The amount of dapsone in the buffer samples was quantified using ultraviolet [10] visible spectroscopy at 293 nm. The release data was fitted to various kinetic models like first order mode (equation 1), zero order model (equation 2), Hixson- Crowell cube root (equation 3), D Higuchi (equation 4) and Korsemeyer-Peppas (equation 5). ! = k# t ------------------------ equation 1 TE ln AC C EP M% − M& = k % t----------------- equation 2 W% #) ( − W& #) ( = k #) t----- equation 3 ( M& = K√t ------------------------- equation 4 , = kt - ------------------------ equation 5 where, W0 and Wt corresponds to the weight of the drug taken initially and at time t, respectively. The terms M0, Mt, and M∞ correspond to the amount of dapsone taken at time equal to zero, dissolved at a particular time (t), and at infinite time, respectively. The terms k1, k0, k1/3, K, and k 6 ACCEPTED MANUSCRIPT represent the release kinetic constants obtained from the linear curves of first-order, zero-order, Hixson-Crowell cube root law, Higuchi model and Korsemeyer–Peppas respectively. The mathematical kinetic modeling was performed using DD Solver (an add-in program for Microsoft Excel) to identify the pattern of drug release from DC. RI PT 2.8 Permeation study across pig ear skin The permeation of DC, marketed formulation and dapsone solubilised in pH 7.4 PBS (dapsonePBS) across freshly excised white pig ear skin was assessed using a Franz diffusion cell [25]. The M AN US C excised pig ear was obtained from a local slaughterhouse. The epidermal layer was carefully excised and washed well to maintain its integrity. The epidermal layer with a surface area of 1.5 cm2 was mounted in the Franz diffusion cell. The receptor compartment was filled with 15 ml of pH 7.4 PBS containing 1% tween 80 as the buffer solution, maintained at 37 ± 1 °C. One ml of DC was applied on the epidermal surface of the pig ear skin and covered with paraffin film to prevent evaporation. Periodically (at 2, 4, 6, 8, 12, 18, and 24 h), buffer solution in the receptor D compartment was withdrawn and immediately replenished with equal volume of fresh buffer TE solution. The amount of dapsone present in the receptor chamber was analyzed by ultravioletvisible spectroscopy at 293 nm. The cumulative percentage of dapsone permeated per unit area EP (µg/cm2) of the pig ear skin was plotted as a function of time (h) and the slope was calculated from the linear portion of the curve. The flux (µg/cm2) at steady state was calculated by dividing the AC C slope by area of the skin surface through which permeation took place. 2.9 Stability studies Stability studies were carried out at 4 ± 1 ºC and 25 ± 1 ºC for a period of 1 year in amber-colored borosilicate glass bottles. Particle size and entrapment efficiency were measured at regular time intervals of 3, 6, 9 and 12 months respectively [26]. 2.10 Statistical analysis 7 ACCEPTED MANUSCRIPT All data were expressed as mean standard deviation (SD) of 3 values. Statistical analysis was performed using one way analysis of variance and the differences between the groups was compared by Bonferroni post-test using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA). A probability value (p) less than 0.05 was considered as statistically significant. RI PT 3. Results and discussion An attempt was made to deliver dapsone across the pig epidermis by encapsulating it in cubosomes. DC were prepared by a simple method based on dispersion, emulsification and hot homogenization M AN US C of an unsaturated monoglyceride and nonionic surfactant. Initially, a clear cubic crystalline gel phase containing dapsone, GMO and poloxamer 407 was formed. Since dapsone is lipophilic, the cubic lipid phase of GMO could able to solubilize and hold dapsone in it. This cubic liquid crystalline gel was converted into particulate dispersion by the application of ultrasonic energy to form cubosomes. Three different macroscopic cubic phases namely precursor gel phase, bulk gel phase, and particulate dispersions (cubosomes) exist at this stage. The precursor form exists either D as solid or liquid state which would form particulate dispersions in aqueous environment. Bulk TE cubic gel phase is an optically isotropic form which would also form cubosomes by sonication [11]. Different batches of DC were prepared by varying the concentrations of GMO and poloxamer 407 AC C EP and their influence on various characteristics of DC was studied (Table 1). Preliminary trials were attempted to choose an optimum amount of GMO and poloxamer 407 for the preparation of colloidally stable cubosomes loaded with dapsone. The results of preliminary trials (not shown) indicated that 2.5%, 3.75%, 5% v/v of GMO as the lipid phase was suitable for the preparation of stable DC. When the amount of GMO was lower than 2.5%, opaque gels were formed instead of transparent gels, possibly due to the existence of hexagonal phase rather than cubic phase. Hence, in all the formulations a minimum of 2.5% of GMO was used in the preparation of cubosomes. The amount of poloxamer 407 used also influenced the properties of 8 ACCEPTED MANUSCRIPT cubosomes. Various concentrations of poloxamer 407 ranging from 0.1- 2.5% were used for the preparation of DC. Lower concentration of poloxamer 407 (< 0.5%) caused phase separation, whereas higher concentration (>1.5%) distorted the shape of cubosomes to spherical lipid nanoparticles. Hence 0.5%- 1.5% of poloxamer 407 was used for the preparation of different RI PT batches of DC. Additionally, the duration of sonication was also optimized. Application of low shear forces will generate cubosomes with an undesirable bigger particle size [10]. On the other hand, although application of high shear forces will reduce the particle size, the high energy M AN US C generated would transform the cubic shaped structures to noncubic structures. Hence, an optimum amount of shear forces should be used for the preparation of cubosomes. Based on the preliminary trails, probe sonication for 15 min with 15 s on cycle and 5 s off cycle was found to be optimum for the preparation of DC in the current study. 3.1 Particle size and PDI Particle size and PDI for the formulations were analyzed by Malvern Zetasizer. The particle size of all the formulations (Table 1) was in the range of 39.4 ± 3.6 nm (DC6) to 231.9 ± 7.1 nm (DC1) the D representative particle size distribution graph is shown in Fig. 1 a. All the formulations, except TE DC1, had a particle size below 100 nm. The particle size of the formulations was dependent on the EP amount of GMO and poloxamer 407 used. When the amount of GMO in the formulations increased from 2.5% to 3.75%, the particle size decreased (Table 1). Increased levels of GMO could have AC C enhanced solubilisation of dapsone and facilitated formation of emulsion with a smaller particle size. But, when the amount of GMO was increased from 3.75 to 5%, the particle size of the cubosomes increased (Table 1). Higher levels of GMO beyond a certain amount (3.5%) could have increased the viscosity and hindered emulsification of the cubosomes resulting in generation of particles with bigger size. A negative correlation between the amount of poloxamer 407 and particle size was observed. When the amount of poloxamer 407 in the formulations increased from 0.5%1.5%, the particle size decreased (Table 1). Higher levels of poloxamer 407 could have reduced the surface tension during emulsification and facilitated in the formation of cubosomes with a reduced 9 ACCEPTED MANUSCRIPT particle size. Of all the formulations prepared, a least particle size (39.4 ± 3.6 nm) was observed for formulation DC6, justifying the previously stated claim that 3.75% of GMO and 1.5% poloxamer 407 facilitates production of cubosomes with least particle size. The PDI of the samples (Table 1) indicated that the formulations were moderately polydisperse. In general, probe sonication does not homogenization may help in preparing monodisperse cubosomes. 3.2 Zeta potential RI PT facilitate production of monodisperse samples. Production of cubosomes using high pressure M AN US C The average zeta potential of DC was in the range of -1.4 ± 0.2 to -2.9 ± 0.5 mV (Table 1) and the representative zeta potential distribution graph is shown in Fig. 1 b.. These values indicated that the cubosomes possessed a neutral zeta potential due to the usage of poloxamer 407 (a non ionic stabilizer) and GMO (amphoteric lipid). The partial negative charge could be attributed to the presence of ionized lipid moieties. Poloxamer 407 would form a coating around the surface of the cubic lipid structures to offer stability against aggregation [3,4]. Hence, despite a neutral zeta 3.3 Morphology of the cubosomes D potential, DC would remain stable against aggregation. TE The morphology of the cubosomes was visualized using SEM and AFM to confirm the formation of EP cubic shaped structures. The micrographs of the cubosomes as visualised using AFM and SEM (in Fig. 2. and Fig. 3. respectively) showed individual particles in nanometric scale with cubic ultra AC C structure. The interaction of GMO and poloxamer 407 with water at controlled temperature with the help of hydrogen bonds facilitated formation of cubic shaped structures, cubosomes [27]. Apart from the cubic shaped structures (21.2%), spherical particles (57.5%) and quassi shaped particles (21.3%) were visible in Fig. 2. The effectiveness of this method to produce cubic shaped structures can be further improved by optimising the amount of surfactant and the duration of sonication. Vesicular structures were not observed during the visualisation under the microscope. The particle size of the DC as observed under Fig. 2 and Fig. 3 is not in consensus with the particle size data obtained using Malvern Zetasizer. This could be attributed to the differences in the working 10 ACCEPTED MANUSCRIPT principle involved in the measurement of particle size. Malvern Zetasizer follows the principle of dynamic light scattering, an intensity based technique, whereas, TEM is a number based technique. 3.4 Entrapment efficiency and in vitro drug release RI PT The entrapment efficiency of DC was in the range of 82.5 ± 2.1to 93.7 ± 3.8% (Table 1) and the pattern of results indicate that the entrapment efficiency was dependent on the amount GMO and poloxamer 407 used during preparation. As the amount of GMO increased from 2.5% to 5%, the M AN US C entrapment efficiency of the formulations was also increased (Table1). Higher levels of GMO could have promoted the solubility of dapsone by providing more room for the incorporation of dapsone in its internal lipid structures. When the amount of poloxamer 407 was increased from 0.5% to 1.5%, the entrapment efficiency of the formulations decreased. Higher amount of poloxamer 407 could have facilitated solubilisation and partitioning of dapsone into the aqueous phase, during transition of cubic gel phase to cubosomes. As stated previously, higher amounts of poloxamer 407 promoted generation of smaller sized particles with a small volume for holding drugs. Thus, to D obtain cubosomes with high entrapment efficiency, it would be beneficial to use a comparatively TE higher amount of GMO (5%) and comparatively lower amount of poloxamer 407 (0.5%). Although EP the entrapment of the dapsone in the cubosomes was fair, it is not necessary to obtain formulations with very high entrapment efficiency as suggested by Verma et al and Rattanapak et al. They have AC C stated that a combination of entrapped and free drug could also enhance the penetration of the drugs across the skin [28,29]. Hence, usage of formulations with a lesser entrapment efficiency for transdermal delivery would suffice the need. Although majority of the formulations had a particle size below 100 nm and considerable entrapment efficiencies, formulation DC5 with a second lowest particle size of 42.7 ± 5.8 nm and lowest PDI of 0.28 ± 0.04 was used for performing in vitro release studies across dialysis membrane and in vitro permeation studies across pig ear skin. 11 ACCEPTED MANUSCRIPT The ability of DC to release the entrapped dapsone was tested by performing in vitro release studies in pH 7.4 PBS containing 1% tween 80 using dialysis bag method. In vitro release profile (Fig. 4.) of dapsone from DC showed a biphasic release pattern over a period of 24 h. During the first hour, a burst release (~ 10%) owing to the unentrapped dapsone was observed. This burst phase was RI PT followed by a sustained release pattern of dapsone from the entrapped cubosomes. A maximum of 82.32 ± 2.28 % drug release was observed at the end of 24 h. Although at each time point, it was replenished with, fresh buffer to maintain the sink conditions, it does not mimic the infinite sink conditions as observed in vivo. Hence, 100% of dapsone may not have been released from the pattern of dapsone from the cubosomes. 3.5 Permeation of DC across pig ear skin M AN US C cubosomal matrix. The correlation coefficient (r2) value (0.9312) indicated a first-order release Permeation profile of DC5, marketed formulation, and dapsone-PBS was studied across pig ear skin using a Franz Diffusion cell. Fig. 5 represents the graph depicting the percentage of dapsone D permeated across the pig ear skin from DC5, marketed formulation, and dapsone-PBS over a period TE of 24 h. Amongst the samples tested, only 37.80 ± 4.90 % of the dapsone permeated across the skin for dapsone-PBS. The marketed formulation showed only 81.20 ± 2.80 % permeation across the EP skin in 24 h. The highest permeation across the skin was observed for formulation DC5, where the AC C amount of dapsone permeated across the skin increased to 89.10 ± 3.70 %. The transdermal flux based on the amount of dapsone permeated across the pig ear skin was also calculated for DC5, marketed formulation and dapsone-PBS. A significantly higher (p value < 0.05) transdermal flux was observed for DC5 (71.28 ± 4.65 µg/cm2/h), followed by marketed formulation (55.28 ± 2.13 µg/cm2/h) and dapsone-PBS (45.44 ± 3.09 µg/cm2/h). Permeation of dapsone across the pig ear skin generally happens in two steps. Initial transfer of dapsone (encapsulated in cubosomes) happens from the aqueous cubosomal dispersion on to the surface of the skin. Finally, DC migrate from the surface of the skin to the receptor chamber 12 ACCEPTED MANUSCRIPT containing buffer solution. The enhanced permeation of dapsone through DC5 could have been possible due to the entrapment of dapsone in the nanometric cubic structured particles (cubosomes) made up of GMO and poloxamer 407. GMO and poloxamer 407, penetration enhancers, could have interacted with the lipids of the skin to form channels which facilitated their permeation [15,30]. RI PT Additionally, a particle size less than 100 nm could have assisted in the transfer through the epidermis. The enhanced concentration of dapsone in the skin may be efficient in treating infection and inflammation at the site of application. M AN US C 3.6 Stability studies The results of the particle size and entrapment efficiency of DC5 over a period of 12 months are mentioned in Table 2. When stored at room temperature over a year, a significant (p< 0.05) increase in particle size from 42.7 ± 5.8 nm to 576.8 ± 21.4 nm was noticed. But, upon storage at refrigerated conditions, DC5 remained stable without any significant change in the particle size after one year (47.2 ± 4.1 nm). The high amount of poloxamer 407 present in the cubosomes could D have stabilized the lipid structures by preventing aggregation. At room temperature, the entrapment TE efficiency of DC5 was also reduced from 89.7 ± 3.5% to 52.5 ± 3.4%. During storage at refrigerated conditions, significant reduction in the entrapment efficiency of the cubosomes was not observed EP (Table 2). At room temperature, cubosomes could have gained energy from heat and light present in the surroundings and enhanced the Brownian motion of the cubosomes. This could have resulted in AC C collision of particles against one another and removal of poloxamer 407 coating. These surfaces devoid of poloxamer 407 coating could have promoted adhesion with other particles to generate deformed particles with bigger size and reduced entrapment efficiency. Thus, storage of cubosomes at refrigerated conditions is a suggested option to avoid aggregation and drug leakage. Alternately, different kinds of carriers for dapsone including polymer-lipid-polymer nanoparticles (145.3- 277 nm) [20], lipid-core nanocapsules (114- 125 nm) [21], chitosan microcapsules (8 µm) [27], solid lipid nanoparticles (300 nm) [32] have been reported. Although cubosomes prepared in 13 ACCEPTED MANUSCRIPT the current work are comparatively smaller than the aforementioned carriers of dapsone, the entrapment efficiency and flux vary due the presence of other excipients in the respective formulations. Hence, it may be too early to predict that cubosomes are superior than the other carriers. RI PT 4. Conclusion DC were prepared by ultrasonication using GMO as lipid carrier and poloxamer 407 as stabilizer. In vitro permeation studies of DC in pig ear skin enhanced the permeation of dapsone in a sustained M AN US C manner when compared to the free form. The enhanced levels of dapsone may improve the therapeutic activity at the local site by reducing systemic side effects. Cubosomes can be used for the topical delivery of other drugs to enhance efficacy. Conflict of interest The authors do not report for any conflict of interest. Acknowledgement The authors would like to thank PSG Sons and Charities for providing all the required facilities for D performing this work. The authors would also like to acknowledge Mohini Organic Pvt ltd., AC C EP TE Mumbai for providing us with gift sample of Glyceryl monooleate (GMO). 14 ACCEPTED MANUSCRIPT REFERENCES [1] M.K. Shah, P. Madan, S. Lin, Preparation, in vitro evaluation and statistical optimization of carvedilol-loaded solid lipid nanoparticles for lymphatic absorption via oral administration, Pharm. Dev. Technol. 19 (2014) 475–485. doi:doi:10.3109/10837450.2013.795169. [2] C.J.H. Porter, N.L. Trevaskis, W.N. Charman, Lipids and lipid-based formulations: doi:10.1038/nrd2197. 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Reis, Design and statistical modeling of mannose-decorated dapsone-containing nanoparticles as a strategy of targeting intestinal M-cells, Int. J. Nanomedicine. 11 (2016) 2601–2617. doi:10.2147/IJN.S104908. 17 ACCEPTED MANUSCRIPT FIGURE CAPTIONS: Fig.1. Particle size distribution (a) and zeta potential graph (b) measured using Malvern zetasizer Fig. 2. Atomic force micrographs of dapsone loaded cubosomes Fig. 3. Scanning electron micrographs of dapsone loaded cubosomes RI PT Fig. 4. In vitro drug release profile of dapsone from dapsone loaded cubosomes and dapsone-PBS Fig. 5. In vitro release profile of dapsone from dapsone loaded cubosomes, marketed formulation and AC C EP TE D M AN US C dapsone-PBS across pig ear skin 18 ACCEPTED MANUSCRIPT LIST OF TABLES: Table 1: Composition and physicochemical characteristics of dapsone loaded cubosomes 0.25 2.5 0.5 DC2 0.25 2.5 1 DC3 0.25 2.5 1.5 DC4 0.25 3.75 0.5 DC5 0.25 3.75 1 DC6 0.25 3.75 1.5 DC7 0.25 5 0.5 DC8 0.25 5 1 DC9 0.25 5 1.5 PDI 231.9 ± 7.1 61.9 ± 5.3 52.3 ± 8.4 78.6 ± 4.5 42.7 ± 5.8 39.4 ± 3.6 65.7 ± 3.9 59.4 ± 9.2 56.6 ± 7.1 0.24 ± 0.05 0.32 ± 0.04 0.41 ± 0.03 0.33 ± 0.07 0.28 ± 0.04 0.53 ± 0.03 0.24 ± 0.04 0.47 ± 0.05 0.51 ± 0.07 Zeta Entrapment Potential efficiency (mV) (%) -1.9 ± 0.1 -1.4 ± 0.2 -2.2 ± 0.5 -2.1 ± 0.9 -1.8 ± 0.5 -2.4 ± 0.6 -2.1 ± 0.4 -2.1 ± 0.7 -2.9 ± 0.5 84.3 ± 2.7 83.6 ± 1.6 82.5 ± 2.1 90.8 ± 1.8 89.7 ± 3.5 88.8 ± 3.7 93.7 ± 3.8 92.6 ± 2.9 90.5 ± 1.8 AC C EP TE D DC1 Particle size (nm) RI PT Poloxamer 407 (%) M AN US C Drug GMO Formulation (%) (%) code 19 ACCEPTED MANUSCRIPT Table 2: Particle size and entrapment efficiency of DC5 over a period of 12 months. Values are expressed as mean ± standard deviation (n=3). Polydispersity index Entrapment efficiency (%) Months Months Months 9 12 42.7 ± 5.8 74.3 ± 2.4 121. 1± 3.3 382. 3± 4.1 576. 8± 21.4 42.7 ± 5.8 41.1 ± 3.2 42.8 ± 2.4 45.3 ± 6.9 47.2 ± 4.1 0 3 6 9 12 0.2 8± 0.0 4 0.2 8± 0.0 4 0.3 4± 0.0 5 0.3 1± 0.0 6 0.38 ± 0.07 0.4 5± 0.0 5 0.3 2± 0.0 5 0.5 7± 0.0 6 0.2 9± 0.0 3 0.34 ± 0.05 0 3 RI PT 6 M AN US C 3 6 9 12 89.7 ± 3.5 79.1 ± 5.6 75.8 ± 2.6 68.3 ± 4.2 52.5 ± 3.4 89.7 ± 3.5 88.1 ± 1.9 86.5 ± 3.4 83.1 ± 2.7 82.9 ± 4.7 TE D 0 EP Room temperat ure (25°C) Refrigera ted temperat ure (3 to 5 °C) Average particle size (nm) AC C Storage Condition 20 ACCEPTED MANUSCRIPT TE D M AN US C RI PT LIST OF FIGURES: EP Fig.1. Particle size distribution graph (a) and zeta potential graph (b) of dapsone loaded cubosomes AC C measured using Malvern Zetasizer 21 M AN US C RI PT ACCEPTED MANUSCRIPT AC C EP TE D Fig. 2. Atomic force micrographs of dapsone loaded cubosomes 22 M AN US C RI PT ACCEPTED MANUSCRIPT AC C EP TE D Fig. 3. Scanning electron micrographs of dapsone loaded cubosomes 23 ACCEPTED MANUSCRIPT 110 DC5 Dapsone-PBS 100 RI PT 80 70 60 50 M AN US C Percentage of drug released (%) 90 40 30 20 0 2 4 6 8 10 TE 0 D 10 12 14 16 18 20 22 24 26 Time (hours) AC C EP Fig. 4. In vitro drug release profile of dapsone from dapsone loaded cubosomes and dapsone-PBS 24 100 90 80 RI PT 70 DC5 Acne dap Dapsone-PBS 60 50 40 30 20 10 0 0 2 4 6 8 10 M AN US C Cummulative percentage of drug released (%) ACCEPTED MANUSCRIPT 12 14 16 18 20 22 24 Time (hours) D Fig. 5. In vitro release profile of dapsone from dapsone loaded cubosomes, marketed formulation and AC C EP TE dapsone-PBS across pig ear skin 25